Figure 4. In vitro plasticity of sorted Th22 cells re-stimulated under Th1, Th17 and Th22 conditions.

Cultures of enriched Th17 and Th22 cells were generated from IL-17eGFP x IL-22tdTomato reporter mice, using optimal polarizing conditions for the first 3 d, and cells sorted on day 4 for Th17 cells (CD4⁺CD44⁺IL-17eGFP⁺) or Th22 cells (CD4⁺CD44⁺IL-17eGFP^-IL-22tdTomato⁺). Purified Th17 and Th22 cells were re-stimulated under Th1, Th17 and Th22 conditions for a further 3 d and the expression of IL-17A, IL-22 and IFN-γ determined. Representative plots of IL-17eGFP and IL-22tdTomato expression in re-stimulated, unfixed Th17 and Th22 cells are shown (A). Representative plots of IL-17eGFP, IL-22tdTomato, and IFN-γ expression in re-stimulated, fixed Th17 and Th22 cells are shown (B). Cell populations in FACS plots are pre-gated on CD4⁺CD44⁺ and viable cells. Quantification of IL-17eGFP⁺, IL-22tdTomato⁺ and IFN-γ⁺ cells in sorted Th17 and Th22 populations re-stimulated under Th0, Th1, Th17 and Th22 conditions (C). Note that paraformaldehyde fixation interferes with fluorescence from the tdTomato protein and likely explains the observed decrease in tdTomato signal following intracellular staining (Figure 4B/C). Error bars represent SEM (n=6 per group from three independent experiments). ***p < 0.001, ****p < 0.0001.